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CHAPTER – 2
MATERIALS AND METHODS
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2. MATERIALS AND METHODS
2.1 Subjects
A total of 105 asthmatic children were included in this study. Children with asthma, who
attended the Out Patient Department of Pediatrics, North Bengal Medical College &
Hospital, were registered for the study. The diagnosis of asthma was made by the
physician based on the medical and clinical history, physical examination, symptoms of
the disease, Peak expiratory flow (PEF) estimation, chest X-ray, etc. The inclusion criteria
for the participating subjects included: i. children diagnosed with asthma, ii. age group of 3
– 12 years, iii. residing in and around Siliguri, the sub-himalayan region of West Bengal,
India.
A total of 110 unrelated individuals were included in the study as controls subjects. In the
selection of control subjects the following criteria were taken into consideration:
i. age, sex and ethnicity matching with asthmatic subjects
ii. no history of lung disease or allergy,
iii. no airways hyperresponsiveness and no upper respiratory infections.
The study was approved by the ‘Human Ethics Committee’, University of North Bengal,
Siliguri, West Bengal, India. The details of the study were explained to the
parents/guardians of the children who participated in this study and an informed consent
was obtained from each parent/guardian.
2.2 Collection of the demographic data & clinical history
The collection of demographic data and clinical history from the participating subjects was
made using the questionnaire. The method of data acquisition is based on the interview of
the parents and/or child. The response given to each question was recorded in the
questionnaire. A sample of the questionnaire is attached in Appendix 2.
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2.3 Estimation of prevalence of asthma in children between 3 to 12 years
For the estimation of the prevalence of asthma in children (3-12 years), the numbers of
asthmatic and non-asthmatic children visiting the OPD were recorded in daily basis for the
period of one year (May 2009 to April 2010).
2.4 Collection of Blood Samples
The clotted blood samples (4-5ml) were taken from each participant by vein puncture
method in the Out Patient Department of Pediatrics, North Bengal Medical College &
Hospital. The collected blood samples were brought to the Cellular Immunology
Laboratory, Department of Zoology, University of North Bengal, under appropriate
conditions for further experiments.
2.4.1 Separation of serum
The blood sample was kept undisturbed for 2-3 hours at room temperature for coagulation.
The blood clot was cut and centrifuged at 2000rpm for 10 minutes to separate the serum.
The separated sera were stored in aliquots at -70°C until further analysis.
2.5 Determination of serum CRP level (Latex Agglutination Test)
The level of CRP was estimated in freshly separated serum samples of asthmatic subjects
using the commercially available ‘IMMUNOSTAT® CRP’ kit (Ranbaxy Fine Chemicals
Ltd., SIDCUL, Haridwar, India). The limitation of detection of the kit was <6 mg/L. The
level of CRP was categorized as elevated (≥6mg/L) and normal (<6mg/L). The positive
reaction with elevated CRP concentration was visible as prominent agglutination. The CRP
test was performed in a total of eighty seven asthmatic subjects. The asthmatics were
divided into two groups viz. ICS naïve and ICS treated based on their treatment regime.
A
Figure 12. Latex agglutinaagglutination reaction is vireaction.
2.6 Determination of tota
Total serum IgE level was
The principle of the metho
anti-IgE antibody on the s
coated on the well. On add
complex is formed. Anoth
enzyme is added which re
biotinylated–antibody com
color is formed which
concentration of the unkno
reference samples with kno
per the manufacturer’s inst
plate reader (Bio-Rad). Th
IU/ml with the intra- a
respectively.
2.7 Determination of seru
The commercially availa
Biotechnology, Inc., Rockf
sera of asthmatic and cont
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B C
ination test for serum CRP level. A. A positivevisible. B & C. Negative reactions where there is
tal serum IgE level
as measured using ELISA kits (Accu-Bind, Mono
thod involves the immobilization of the biotinyl
surface of a microplate well on interaction with
ddition of serum containing the native antigen, a
other antibody (directed to a different epitope)
results in the formation of an enzyme labeled an
mplex on the surface of the wells. On addition
h is measured using a microplate spectrop
nown samples is determined from the standard cur
nown antigen concentration. The assay procedure
struction. The absorbance was measured at 450 n
he sensitivity of the IgE AccuBindTM ELISA tes
and inter-assay precisions of 1.95–5.87% a
rum levels of IL-4 and IFN-γ
ilable 96 well ELISA kits (Endogen Human
kford) were used to determine the level of IL-4
ntrol subjects. The sensitivity of the kits was <2
ive reaction where is no agglutination
nobind Inc., USA).
ylated monoclonal
ith the streptavidin
, antibody–antigen
e) labeled with an
antibody–antigen–
on of the substrate
ophotometer. The
curve created using
re was followed as
0 nm in the ELISA
est system was 1.0
and 3.52–8.42%,
an IL kit, Pierce
4 and IFN-γ in the
2 pg/ml with inter
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and intra-assay coefficient of variation of <10% in each case. The absorbance was
measured at 450 nm in a microtitre plate reader (Opsys MR, Dynex Tehnologies).
2.7.1 Assay Procedure
1. 50µl of reconstituted standards and test samples were added to each well.
2. 50µl of Biotinylated Antibody Reagent was added to each well and mixed thoroughly
by gently tapping the plate several times.
3. The plate was covered with an adhesive microtiter plate cover, ensuring all edges and
strips are tightly sealed and the plate was incubated for two hours at room
temperature, 20-25°C.
4. The adhesive plate cover was removed carefully and the plate was washed three times
with wash buffer.
5. 100µl of prepared Streptavidin-HRP Solution was added to each well.
6. A new adhesive plate cover was attached, ensuring all edges and strips are tightly
sealed. The plate was incubated for 30 minutes at room temperature, 20-25°C.
7. The adhesive plate cover was removed carefully, the plate contents were discarded.
Then the plate was washed three times.
8. 100µl of TMB Substrate Solution was added into each well.
9. The plate was allowed to develop color reaction at room temperature in dark for 30
minutes.
10. After 30 minutes, the reaction was stopped by adding 100µl of Stop Solution to each
well. The substrate reaction yielded a blue solution that turned yellow when stop
solution was added.
11. The absorbance was measured on an ELISA plate reader set at 450nm within 30
minutes of stopping the reaction.
2.8 Extraction of genomic DNA
The genomic DNA was extracted from the frozen peripheral blood samples using phenol
chloroform extraction method as described by Comey et al. (1994) with minor
modifications. The procedure is described below:
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1. Blood samples and all the reagents were thawed 30 minutes prior to use.
2. 500µl of blood sample was taken into a 2ml microfuge tube and equal volume of
Red Cell Lysis Buffer was added. Mixed and centrifuged at 5000rpm for 5 minutes.
3. The supernatant was discarded and 1ml of 1X SSC was added. Mixed thoroughly
and centrifuged at 5000rpm for 5 minutes.
4. The supernatant was discarded and 1.2ml of 1X SSC was added. Mixed and
centrifuged at 5000rpm for 3 minutes.
5. The pellet was resuspended with 1.2ml of 50mM KCl and centrifuged as above.
6. The supernatant was discarded and 375µl of High Salt Buffer, 25µl 10% SDS and
12.5µl of 8mg/ml Proteinase K were added. Mixed gently and incubated at 56°C
for 1 hour.
7. Equal volume of Phenol-Chlorofom was added to the PK digested suspension and
spinned at 12000rpm for 20 minutes at 4 °C.
8. The DNA was precipitated in chilled absolute alcohol.
9. The DNA was rinsed with 70% ethanol, twice.
10. The DNA was dried and dissolved in 50µl TE.
2.8.1 Quantification of DNA
10µl of dissolved DNA was diluted to 1.5ml using deionized water and mixed properly.
The OD was measured at wavelengths of 260nm and 280nm in a UV spectrophotometer.
The DNA samples with 260/280 ratio of 1.7 and above were free of protein
contaminations. The concentration of DNA was calculated using the formula:
OD at 260nm X dilution factor X 50 (1OD = 50µg of double stranded DNA).
The purity and integrity of DNA samples were also checked in 1% pre-stained agarose gel
(Figure 13).
Figure 13. Electrophoregra
after extraction.
2.9 PCR-SSP Typing of H
Molecular typing of the sel
polymerase chain reaction
sequences of the primers o
and Table 6, respectively.
from Bunce et al. (1995) a
and Sacchetti et al. (1997).
2.9.1 PCR amplification
A 25µl reaction mix was p
MiniTM Gradient Thermal
controls (hemoglobin gene
growth hormone gene1 of
amplification.
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gram showing DNA samples run in 1% pre-stai
HLA alleles
selected HLA class I and class II allelic groups w
on using sequence-specific primers (PCR-SSP).
s of HLA class I and class II allelic groups are s
y. The primer sequences of class I allelic group
) and of class II allelic groups were taken from Z
.
prepared and the reaction was carried out in a the
al Cycler, PTC-1148, Bio-Rad, Singapore). Two
ne of 256bp for class I allelic groups and a frag
f 439bp for class II allelic groups) were used to m
tained agarose gel
was performed by
P). The nucleotide
shown in Table 5
ups were obtained
Zhu et al. (2007)
thermal cycler (MJ
o different internal
ragment of human
o monitor the PCR
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Table 5. Primer sequences of HLA class I allelic groups
Allelic group Sequence No. of bases
HLA-A*01 5’-GGACCAGGAGACACGGAATA-3’ 20
5’-AGG TAT CTG CGG AGC CCG-3’ 18
A*03 5’-AGC GAC GCC GCG AGC CA-3’ 17
5’-CAC TCC ACG CAC GTG CCA-3’ 18
A*11 5’-ACG GAA TGT GAA GGC CCA G-3’ 19
5’- CTC TCT GCT GCT CCG CCG-3’ 18
A*24 5’-GGC CGG AGT ATT GGG ACG A-3’ 19
5’-CCT CCA GGT AGG CTC TCT G-3’ 19
A*25 5’-TCA CAG ACT GAC CGA GAG AG-3’ 20
5’-ATG TAA TCC TTG CCG TCG TAA-3’ 21
A*26 5’-ACT CAC AGA CTG ACC GAG C-3’ 19
5’-ATG TAA TCC TTG CCG TCG TAA-3’ 21
B*08 5’-GAC CGG AAC ACA CAG ATC TT-3’ 20
5’-CCG CGC GCT CCA GCG TG-3’ 17
B*37 5’-GCC GCG AGT CCG AGG AC-3’ 17
5’-CCT CCA GGT AGG CTC TGT C-3’ 19
B*44 5’-TAC CGA GAG AAC CTG CGC-3’ 18
5’-CCA GGT ATC TGC GGA GCG-3’ 18
B*45 5’-ACC GGG AGA CAC AGA TCT C-3’ 19
5’-CCA GGT ATC TGC GGA GCG-3’ 18
B*51 5’-GGA GTA TTG GGA CCG GAA C-3’ 19
5’-CGT TCA GGG CGA TGT AAT CT-3’ 20
B*52 5’-ACCGGGAGACACAGATCTC-3’ 19
5’-CGT TCA GGG GGA TGT AATCT-3’ 20
Hemoglobin 5’-ATG ATG TTG ACC TTT CCA GGG-3’ 21
gene (PIC-1) 5’-ATT CTG TAA CTT TTC ATC AGT TGC-3’ 24
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Table 6. Primer sequences of HLA class II allelic groups
Allelic group Specific sequence No. of bases
HLA- DRB1*03 5’ GTTTCTTGGAGTACTCTAGGTC 3’ 22
5’ TGCAGTAGTTGTCCACCCG 3’ 19
HLA-DRB1*04 5’ GTTTCTTGGAGCAGGTTAAACA 3’ 22
5’ CGCTGCACTGTGAAGCTCTC 3’ 20
HLA-DRB1*12 5’ AGTACTCTACGGGTGAGTGTT 3’ 21
5’ CTGTTCCAGGACTCGGCGA 3’ 19
HLA-DRB1*01 5’ TTGTGGCAGCTTAAGTTTGAAT 3’ 22
5’ GCTGTTCCAGTACTCGGCAT 3’ 20
HLA-DQB1*0201 5’ GTGCGTATTGTGAGCAGAAG 3’ 20
5’GCAAGGTCGTGCGGAGCT 3’ 18
HLA-DQB1*0302 5’ GACGGAGCGCGTGCGTCT 3’ 18
5’ AGTACTCGGCGTCAGGCG 3’ 18
HLA-DQB1*0603/8 5’ GGAGCGCGTGCGTCTTGTA 3’ 19
5’ GCTGTTCCAGTACTCGGCAT 3’ 20
HLA-DQA1*0501 5’ AGCAGTTCTACGTGGACCTGGGG 3’ 23
5’ GGTAGAGTTGGAGCGTTTAATCAGA 3’ 25
Human GH gene 5’ CAGTGCCTTCCCAACCATTCCCTTA 3’ 25
(PIC-2) 5’ ATCCACTCACGGATTTCTGTTGTGTTTC 3’ 28
2.9.2 Preparation of reaction mixture
The reaction mix of 25µl was prepared in the following proportions:
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Table 7. Preparation of reaction mixture for PCR
Reaction components Concentration Reaction mixture
MilliQ water Taq Buffer dNTPs 100mM Primer (Forward) Primer (Reverse) PIC-F PIC-R Taq Pol Template
- Containing 15 mM MgCl2 10 mM 10 pm 10 pm 10 pm 10 pm 3U/ µl 50 µg
13 µl 2.5 µl 3.0 µl 1.5 µl 1.5 µl 0.6 µl 0.6 µl 0.3 µl 2.0 µl
Volume 25 µl
2.9.3 Amplification procedure
A 25µl reaction mix was dispensed into PCR tubes. The tubes were spun for 1 min for
mixing up of reaction components uniformly and the tubes were lodged into the thermal
cycler. The touch down method was adopted for the PCR amplification. The reaction
condition that was followed for PCR amplification is shown in Table 8.
Table 8. Reaction conditions followed for DNA amplification
No. of cycle Time Temperature
1 cycle of denaturation 3 min 94°C 5 cycles:
Denaturation 30 s 94°C Annealing 35 s annealing*+2°C Extension 40s 72°C 25 cycles:
Denaturation 30s 94°C Annealing 50s annealing*-2°C Extension 1min 72°C 1 cycle of extension 7min 72°C Hold Forever 12°C *
Annealing temperature varying for different alleles
2.9.4 Amplification check
Mini gel electrophoresis ap
amplified PCR products. T
Sisco Research Laboratorie
Mannheim, Germany) in T
The step up 100bp DNA m
of uniform intensity (1000
100bp) was loaded in one o
dye (Bromophenol blue an
carried out at 80V for 1hr
allelic groups are shown in
A. HLA-A*11 (518 bp)
C. HLA-B*52 (440 bp)
E. HLA-DQB1*0201 (205 bp
Figure 14. Electrophoregraclass II allelic groups run internal control (256 bp) &M: 100 bp DNA ladder (105: A*24 (555bp), C. Lane 21, 4 & 8: DQB1*0201 (205
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ck by agarose gel electrophoresis
apparatus (BIOTECH, India) was used for the ra
The PCR products were separated on 2% agar
ries, India) gel containing 0.5µg/ml ethidium brom
TBE buffer to check efficiency and specificity
marker (Banglore Genie, India) providing even
00bp, 900bp, 800bp, 700bp, 600bp, 500bp, 400bp
e of the wells. 10-12 µl of PCR products were mi
and sucrose) and loaded into the wells. The ele
hr to 1hr 30 minutes. Amplified PCR products
in Figure 14.
B. HLA-A*24 (555 bp)
D. HLA-DRB1*12 (163 bp)
bp) F. HLA-DQA1*0501 (144 bp)
ram showing amplified PCR products of various n in 2% pre-stained agarose gel. Lane 1-8 in A, & Lane 1-8 in D, E & F: Positive internal contro100 bp to 1000 bp). A. Lane 3 & 5: A*11 (518bp)2 & 5: B*52 (440bp), D. Lane 2 & 5: DRB1*12
05bp) and F. Lane 2, 5 & 6: DQA1*0501 (144bp).
rapid separation of
garose (Low EEO,
omide (Boehringer
ty of the reaction.
n banding patterns
bp, 300bp, 200bp,
ixed with loading
lectrophoresis was
ts of various HLA
us HLA class I and , B & C: Positive
trol (439 bp). Lane p), B. Lane 1, 4 & 2 (163bp), E. Lane p).
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2.9.4.1 Procedure
Agarose gels (2%) were prepared by suspending 1g dry agarose in 50ml 1X TBE buffer,
then boiling the mixture until a clear solution was formed. This was allowed to cool down,
8-10µl of 0.5µg/ml ethidium bromide was added and mixed thoroughly by swirling and the
content was poured in the gel cast sealed with tape on both sides. The comb was placed
and it was allowed to form a rigid gel at room temperature.
2.9.4.2 Documentation and Interpretation
The gel was visualized under the UV-transilluminator (Vilber Laurmat, France) and the
photographic documentation of the gel was done using Polaroid camera and analyzed
using BioID Analysis software (Vilber Laurmat, France).
2.10 Statistical Analysis
Data were statistically analyzed using SPSS, version 16. Mean and standard deviation were
calculated for the variables and t-test was employed for the comparisons. For the
attributes, percentages were calculated and χ2 test was used for comparisons. Figures were
drawn using OriginLab 6.1. The frequencies of HLA allelic groups and HLA A-B
haplotypes were calculated by direct counting (Mathews, 1984). The frequency of each
allele and haplotype observed in the asthmatic group was compared to control group using
χ2 test. P-value was corrected (pcorr) for multiple comparisons by multiplying with the
number of allelic groups. A p-value<0.05 was considered statistically significant. The odds
ratio (OR) and the 95% CI for each HLA allele were calculated using the GRAPHPAD
INSTAT version 3.10.